The present invention relates generally to the enzyme-catalyzed modification of insoluble macromolecules in organic solvents.
Selective modification of macromolecules is desirable to tailor their structural and functional properties, such as hydrophobicity, hydrophilicity, and interfacial and film forming properties. E.g., fatty acid esters of macromolecules may be useful as bioerodable and biodegradable drug delivery matrices such as coatings, finishes and films, and biodegradable emulsifiers, compatibilizers and detergents.1 However, selective acylation of hydroxylated macromolecules by chemical reactions are difficult due to multiple steps involved in the modification (protection, deprotection, extraction, separation and purification), and due to the lack of specificity, solubility and the presence of multifunctional groups in the polymer.2 Enzymes have been used to acylate saccharides of different glucose moieties regioselectively under mild condition in organic solvents. 2-4 Similar reactions with other hydroxylated macromolecules would be desirable. However, the lack of solubility of these polymers and the enzymes in organic solvents implies significant problems in carrying out these conversions. Accordingly, alternate methods of achieving a functionally significant degree of modifications are required.5-7
Enzymes are powerful catalysts in organic solvents where they catalyze a wide variety of reactions that are difficult to perform in aqueous solutions. This is particularly evident in esterification reactions catalyzed by lipases and proteases wherein a variety of nucleophiles act as substrates for enzyme-catalyzed acyl transfer in nearly anhydrous organic solvents. Unfortunately, many hydroxylated compounds are either sparingly soluble in only the most polar organic solvents, or are completely insoluble in organic media. For these substrates, conventional non-aqueous enzymology is unable to support catalytic transformations. The development of a suitable technique for the selective modification of macromolecules in organic solvents, therefore, would represent both an opportunity for the synthesis of novel materials as well as means to overcome a technical hurdle in the broader uses of enzymes in non-aqueous media.
Accordingly, it is an object of this invention to overcome the above illustrated inadequacies and problems of insoluble macromolecules by providing a improved method of their modification.
It is another object of this invention to provide a method of acylating hydroxylated macromolecules wherein their selective modification results in structural and/or functional benefits.
Yet another object of the present invention is to provide a method of enabling the use of enzymes to catalyze reactions in non-aqueous media for the synthesis of biodegradable, bioerodable and biocompatible compounds.
The present invention provides process for the production of esterified polyhydroxylated macromolecules such as proteins, where the esterification is limited to alcohols of serine, threonine and hydroxyproline present at the surface of the proteins. Such attributes are novel because chemical esterification is unable to modify selectively such macromolecules. The processes also emphasize the use of mild reaction conditions such as temperature, pressure and pH, recycling of the reaction media leading to waste minimization, minimal by product formation, and minimal separations and purifications. Reaction media and the catalysts can be easily regenerated to minimal product cost and environmental hazard. Since the reactions are carried out at ambient conditions and selectively, the biological activity of the macromolecules such as enzyme activity and binding affinity are retained even after esterification.
Recently, a method was developed to solubilize enzymes in hydrophobic organic solvents through the formation of enzyme-surfactant ion pairs.8 These ion-paired, organic-soluble enzymes are extremely active in hydrophobic solvents, such as isooctane. The present invention demonstrates that hydroxylated macromolecules such as silk proteins, collagen, xanthan gum, hyaluronic acid, polyvinyl alcohol and polyethylene glycol when deposited as thin film or cryogenically milled can be selectively acylated by catalysis in organic solvents using an organic-soluble enzyme preparation of subtilisin (from Bacillus subtilis). This represents the first attempt at catalyzing solvent-insoluble macromolecular modification using enzymes in organic solvents.
Proteases such as subtilisin (from Bacillus subtilis), ion paired with AOT, remained predominantly active and soluble in isooctane, To enhance the reactivity of insoluble substrates, thin layers and cryogenically milled powders of hydroxylated macromolecules such as silk proteins, collagen, xanthan gum hyaluronic acid, polyvinyl alcohol and polyethylene glycol were prepared to increase the surface area and some of these biopolymers and synthetic polymers were selectively esterified using ion-paired proteases.
Modified macromolecules can be used for biodegradable emulsifiers, bioerodable films, coatings and fibers, hydrogels, compatibilizers and detergents. Of particular importance is the potential use of these low cost polymers for edible wrapping films utilized for food storage. In addition to the above applications, esterified macromolecules can be of extreme interest to the paper industry in order to efficiently recycle paper and other compounds based on cellulose. Since the reactions are carried out selectively using mild conditions, biological activities of these macromolecules may be enhanced by changing their transport and binding affinities. Another application that can be envisioned in the manufacturing of drug delivery systems as hydrogels (pH, temperature and pressure sensitive), specific filters, high absorbance compounds, coatings, finishes, films, fibers and membranes.
Other objects, features and advantages will be apparent from the following detailed description of preferred embodiments thereof taken in conjunction with the accompanying drawings.